DESCRIPTION (Applicant's Abstract): Salmonella is one of the most extensively characterized bacterial pathogens and is a leading cause of bacterial gastroenteritis. With the aging of the population we are likely to see further increases in the incidence of this infection, and we can expect an increase in the severity of disease because the elderly are more susceptible to disease and tend to have more severe infections. It is clear that the SPI1 encoded type III secretion apparatus and the proteins secreted by this apparatus play a major role for invasion of the epithelium, recruitment of PMNs, and diarrhea. A better understanding of this system and its role in pathogenesis of diseases caused by Salmonella should lead to better methods of treatment and prevention. We recently identified the sigDE locus of S. typhimurium; sigD encodes an effector protein secreted by the SPI1 type III secretion apparatus (note: effector proteins are those believed to have a direct effect on host cells). Like other effectors in this system, expression of sigDE requires SirA and HilA, but as with other effectors, expression of sigDE is probably not directly affected by these regulators. The current model suggests that SirA turns on expression of HilA which turns on expression of invF. InvF is thought to activate expression of genes encoding the effectors; we have demonstrated that InvF is required for sigD expression. In addition, a screen for regulators of sigD identified SicA (encoded by SPI1). SicA is thought to be a chaperone for SipB and SipC. How expression of the type III secretion system and expression of the translocators and effector proteins is coordinated remains an important question. These are the questions we hope to address in this proposal with the long-term goal of understanding how these events are integrated with other virulence factors of Salmonella to cause disease. Specifically we propose the following: Aim 1. Which transcriptional units are regulated by InvF and SicA? To test this we will identify the key transcriptional units and will examine expression of reporter constructs in different mutant backgrounds. Aim 2. How do SicA and InvF mediate regulation of the sigDE and other promoters? To do this we will examine the interactions of SicA and InvF with each other and the promoters they regulate. Aim 3. To identify other effectors secreted by the SPI1 type III secretion pathway by identifying InvF regulated genes. These will be characterized for their role in virulence related assays.